The study investigated the specific mechanism by which oxocrebanine, the anti-hepatic cancer active ingredient in Stephania hainanensis, inhibits the proliferation of hepatic cancer cells. Firstly, methyl thiazolyl tetrazolium(MTT) assay, 5-bromodeoxyuridine(BrdU) labeling, and colony formation assay were employed to investigate whether oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells. Propidium iodide(PI) staining was used to observe the oxocrebanine-induced apoptosis of HepG2 and Hep3B2.1-7 cells. Western blot was employed to verify whether apoptotic effector proteins, such as cleaved cysteinyl aspartate-specific protease 3(c-caspase-3), poly(ADP-ribose) polymerase 1(PARP1), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), Bcl-2 homologous killer(Bak), and myeloid cell leukemia-1(Mcl-1) were involved in apoptosis. Secondly, HepG2 cells were simultaneously treated with oxocrebanine and the autophagy inhibitor 3-methyladenine(3-MA), and the changes in the autophagy marker LC3 and autophagy-related proteins [eukaryotic translation initiation factor 4E-binding protein 1(4EBP1), phosphorylated 4EBP1(p-4EBP1), 70-kDa ribosomal protein S6 kinase(P70S6K), and phosphorylated P70S6K(p-P70S6K)] were determined. The results of MTT assay, BrdU labeling, and colony formation assay showed that oxocrebanine inhibited the proliferation of HepG2 and Hep3B2.1-7 cells in a dose-dependent manner. The results of flow cytometry suggested that the apoptosis rate of HepG2 and Hep3B2.1-7 cells increased after treatment with oxocrebanine. Western blot results showed that the protein levels of c-caspase-3, Bax, and Bak were up-regulated and those of PARP1, Bcl-2, and Mcl-1 were down-regulated in the HepG2 cells treated with oxocrebanine. The results indicated that oxocrebanine induced apoptosis, thereby inhibiting the proliferation of hepatic cancer cells. The inhibition of HepG2 cell proliferation by oxocrebanine may be related to the induction of protective autophagy in hepatocellular carcinoma cells. Oxocrebanine still promoted the conversion of LC3-Ⅰ to LC3-Ⅱ, reduced the phosphorylation levels of 4EBP1 and P70S6K, which can be reversed by the autophagy inhibitor 3-MA. It is prompted that oxocrebanine can inhibit the proliferation of hepatic cancer cells by inducing autophagy. In conclusion, oxocrebanine inhibits the proliferation of hepatic cancer cells by inducing apoptosis and autophagy.
Humans ; Apoptosis/drug effects* ; Autophagy/drug effects* ; Cell Proliferation/drug effects* ; Hep G2 Cells ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/genetics* ; Caspase 3/genetics*

Compounds isolated from Epimedium include the total flavonoids of Epimedium , icariin, and its metabolites (icaritin, icariside I, and icariside II), which have similar molecular structures. Modern pharmacological research and clinical practice have proved that Epimedium and its active components have a wide range of pharmacological effects, especially in improving sexual function, hormone regulation, anti-osteoporosis, immune function regulation, anti-oxidation, and anti-tumor activity. To date, we still need a comprehensive source of knowledge about the pharmacological effects of Epimedium and its bioactive compounds on the male reproductive system. However, their actions in other tissues have been reviewed in recent years. This review critically focuses on the Epimedium , its bioactive compounds, and the biochemical and molecular mechanisms that modulate vital pathways associated with the male reproductive system. Such intrinsic knowledge will significantly further studies on the Epimedium and its bioactive compounds that protect the male reproductive system and provide some guidances for clinical treatment of related male reproductive disorders.
Male ; Epimedium/chemistry* ; Humans ; Genitalia, Male/drug effects* ; Flavonoids/therapeutic use* ; Animals

OBJECTIVE: To evaluate the dynamic changes of glucocorticoid (GC) dose and the feasibility of GC discontinuation in rheumatoid arthritis (RA) patients under the background of Chinese medicine (CM). METHODS: This multicenter retrospective cohort study included 1,196 RA patients enrolled in the China Rheumatoid Arthritis Registry of Patients with Chinese Medicine (CERTAIN) from September 1, 2019 to December 4, 2023, who initiated GC therapy. Participants were divided into the Western medicine (WM) and integrative medicine (IM, combination of CM and WM) groups based on medication regimen. Follow-up was performed at least every 3 months to assess dynamic changes in GC dose. Changes in GC dose were analyzed by generalized estimator equation, the probability of GC discontinuation was assessed using Kaplan-Meier curve, and predictors of GC discontinuation were analyzed by Cox regression. Patients with <12 months of follow-up were excluded for the sensitivity analysis. RESULTS: Among 1,196 patients (85.4% female; median age 56.4 years), 880 (73.6%) received IM. Over a median 12-month follow-up, 34.3% (410 cases) discontinued GC, with significantly higher rates in the IM group (40.8% vs. 16.1% in WM; P<0.05). GC dose declined progressively, with IM patients demonstrating faster reductions (median 3.75 mg vs. 5.00 mg in WM at 12 months; P<0.05). Multivariate Cox analysis identified age <60 years [P<0.001, hazard ratios (HR)=2.142, 95% confidence interval (CI): 1.523-3.012], IM therapy (P=0.001, HR=2.175, 95% CI: 1.369-3.456), baseline GC dose ⩽7.5 mg (P=0.003, HR=1.637, 95% CI: 1.177-2.275), and absence of non-steroidal anti-inflammatory drugs use (P=0.001, HR=2.546, 95% CI: 1.432-4.527) as significant predictors of GC discontinuation. Sensitivity analysis (545 cases) confirmed these findings. CONCLUSIONS RA patients receiving CM face difficulties in following guideline-recommended GC discontinuation protocols. IM can promote GC discontinuation and is a promising strategy to reduce GC dependency in RA management. (Trial registration: ClinicalTrials.gov, No. NCT05219214).
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Arthritis, Rheumatoid/drug therapy* ; Glucocorticoids/therapeutic use* ; Medicine, Chinese Traditional ; Retrospective Studies

The paper is to establish an UPLC-MS/MS method for the simultaneous determination of 19 components in Microctis Folium from different production areas. The 50% methanol was used as extraction solvent. The Agilent ZORBAX SB C18 (150 mm × 2.1 mm, 1.8 μm) column was used; mobile phase was acetonitrile - 0.1% acetic acid with gradient elution, flow rate was 0.3 mL·min^-1, colume temperature was 30 ℃, and the injection volume was 2 μL; electrospray ionizaton source was used and detected in negative ion mode. The results showed that the established UPLC-MS/MS method could well separate the 19 components, and the methodological investigation results of 19 components were good. By means of orthogonal partial least squares discriminant analysis (OPLS-DA), 28 batches of Microctis Folium samples from different production areas can be divided into three categories, Guangdong, Guangxi and Hainan are each classified into one category, and 10 signature compounds which affecting the quality differences of different production areas were screened out. The established method is accurate, reliable, sensitive and reproducible. It can provide a basis for the establishment of the quality standard of Microctis Folium, as well as for safety and quality research.

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.
Chromones ; Sodium Chloride ; Chromatography, Liquid ; Flavonoids ; Tandem Mass Spectrometry ; Thymelaeaceae

Cytochrome P450 reductase (CPR) is essential for the electron transport chain of cytochrome P450s, playing an indispensable role in electron transfer in vivo. In this study, one cDNA encoding cytochrome P450 reductase (Ascpr1) was identified from the callus of Aquilaria sinensis. Ascpr1 contains an open reading frame of 2 124 bp. The deduced protein is composed of 707 amino acids, with a predicted molecular weight of 78.82 kD. Phylogenetic analysis revealed that AsCPR1 is a type Ⅱ CPR protein closely related to the CPR from Theobroma cacao. Transmembrane prediction using TMHMM 2.0 indicated that the amino acids 52-71 of AsCPR1 comprise a transmembrane region. After truncating of 67 amino acid residues from N-terminal, the truncated AsCPR1 was successfully expressed in E. coli Transetta (DE3). Further purification of the recombinant AsCPR1 by affinity chromatography and determination of the enzymatic activity allowed the reducing ability of AsCPR1 to cytochrome C in vitro. The results pave the way for further study on the synthesis of defensive chemicals involved in P450s and the functions of CPR in self-defense of A. sinensis.

Lysine decarboxylase is a key enzyme involved in the upstream biosynthesis of lycopodium alkaloids (LAs) such as huperzine A, contributing to the decarboxylation of lysine to 1,5-pentanediamine (cadaverine). Three lysine decarboxylase genes (HsLDC-L1, HsLDC-L2, HsLDC-L3) were successfully cloned from Huperzia serrata using transcriptomic sequence data mining strategy combined with reverse transcription PCR. The physicochemical properties, secondary and tertiary structures, amino acid identities, and evolutionary relationship of the three LDCs were analyzed by online bioinformatics analysis platforms and DNAMAN, MEGA 7.0 software, revealing that all of these proteins had the conserved PLP binding domain and active site residues were completely conserved in LDCs. Phylogenetic analysis showed that these LDCs were located in the same branch as other known LDCs from LA-producing plants. Accordingly, the ORFs of these three HsLDCs were inserted into different expression plasmids for further expression in E. coli. However, only HsLDC-L1 was successfully expressed in E. coli BL21 (DE3) by inserting into a pCold TF vector. The recombinant protein was purified by Ni²⁺ affinity chromatography purification. HsLDC-L1 contains 469 amino acid residues, with a calculated molecular weight of 50.50 kDa. HsLDC-L1 expectedly catalyzed the decarboxylation of lysine to produce cadaverine. In addition, HsLDC-L1 can also catalyze the generation of putrescine from ornithine. However, it cannot catalyze the decarboxylation of tyrosine, phenylalanine, tryptophan and histidine. The results not only provide insight into the biosynthesis of LAs including huperzine A, but also provide a critical genetic element for the overproduction of Δ¹-piperideine and pelletierine, the essential biosynthetic precursors of LAs, using synthetic biology strategies.

BACKGROUND: Extracellular vesicles (EVs) are derived from internal cellular compartments, and have potential as a diagnostic and therapeutic tool in degenerative disease associated with aging. Mesenchymal stem cells (MSCs) have become a promising tool for functional EVs production. This study investigated the efficacy of EVs and its effect on differentiation capacity. METHODS: The characteristics of MSCs were evaluated by flow cytometry and stem cell differentiation analysis, and a production mode of functional EVs was scaled from MSCs. The concentration and size of EVs were quantitated by Nanoparticle Tracking Analysis (NTA). Western blot analysis was used to assess the protein expression of exosomespecific markers. The effects of MSC-derived EVs were assessed by chondrogenic and adipogenic differentiation analyses and histological observation. RESULTS The range of the particle size of adipose-derived stem cells (ADSCs)- and Wharton’s jelly -MSCs-derived EVs were from 130 to 150 nm as measured by NTA, which showed positive expression of exosomal markers. The chondrogenic induction ability was weakened in the absence of EVs in vitro. Interestingly, after EV administration, type II collagen, a major component in the cartilage extracellular matrix, was upregulated compared to the EV-free condition.Moreover, EVs decreased the lipid accumulation rate during adipogenic induction.

The normal function of vascular endothelial cells is an important foundation for maintaining vascular permeability， restricting inflammatory activities of the vascular wall， and balancing the coagulation and fibrinolytic system. Endothelial dysfunction caused by persistent damage from pathological factors is considered as an early and prominent event of diabetic macroangiopathy. In traditional Chinese medicine， the classical theory of "restraining excessiveness to acquire harmony" was originally a condensed generalization of the rule of generation， restriction， replacement and evolution in the natural world， revealing the internal regulation mechanism of the stable operation of things. Then it gradually evolved into an important rule to explain the physiological phenomena and pathological mechanism of human body and guide the treatment. Corresponding to the nature， the body homeostasis also requires to achieve a state of strong viscera function and inexhaustible Qi and blood generation under the rule of restriction and generation. The pathogenesis of diabetic macroangiopathy is the process of "the predominant one failing to restrict and the hyperactive one becoming harmful". The loss of restriction and generation of the five organs leads to powerless Qi transformation and， as a result， the Qi， blood and body fluid cannot be dispersed. Therefore， the Qi， blood and body fluid turn into phlegm and blood stasis， such as glucose and lipid metabolism disorder， oxidative stress， inflammatory response and high blood viscosity， and finally block the veins. Excessive phlegm and blood stasis cannot be resolved， instead they become harmful and invade the blood vessel， causing endothelial dysfunction and further resulting in diabetic macroangiopathy. Under the guidance of the theory of "restraining excessiveness to acquire harmony"， the method of "harmonizing viscera， eliminating pathogen and removing turbidity" can effectively regulate the function of vascular endothelial cells， thus playing a positive role in preventing and treating diabetic macroangiopathy. The mechanism may be related to reducing oxidative stress， inhibiting inflammation， limiting vascular smooth muscle proliferation， and reducing platelet adhesion.